Protein Interactions

Question 1

List some classes of DNA binding proteins

Answer 1

Leucine zipper motifs
Zinc fingers
Basic helix loop helix
B-sheet

Question 2

Where do most DNA binding domains interact with DNA?

Answer 2

With the major groove

Question 3

Describe the basic properties of Leucine Zippers

Answer 3

Two alpha helices binding domains
Form an inverted Y structure where each arm mediates binding to a specific DNA sequence in the major groove of DNA (TGTT)
Each alpha helix bind to one hald of symetric DNA structure

Question 4

Describe the basic properties of Zinc Fingers

Answer 4

Recognises DNA by using the 3 zinc fingers of cys-cys-his-his type repeats
The three fingers have similar amino acid sequences
The zinc finger holds together an alpha helix and beta sheet

Question 5

Describe the basic properties of basic helix loop helix

Answer 5

Two monomers are held together in a four helix bundle: each monomer contributes two alpha helixes connected by a flexible loop of protein
A specific DNA sequence is bound by two alpha helixes that project from the four helix bundle

Question 6

Give three examples where metal is bound in proteins

Answer 6

Structural eg in zinc finger
Regulatory eg in calmodulin
Catalytic eg zinc, iron and copper

Question 7

How is zinc coordinated in the zinc finger proteins?

Answer 7

Tetrahedrally

Usually by cysteine or histidine residues - these proteins grab the zinc

Question 8

At which position in the zinc finger protein can you find the cysteine and histeine residues?

Answer 8

Cys = 3 and 6
His = 19 and 23

Question 9

Each zinc finger recognises how many nucleotides?

Answer 9

2

Question 10

Which assay is used to study the strength and specificity of a proteins interaction with different DNA sequences?

Answer 10

Gel-mobility shift assay

Question 11

What is the purpose of DNA footprinting?

Answer 11

Determines the DNA sequence recognised by a gene regulatory protein once it has been purified

Question 12

How does DNA footprinting work?

Answer 12

It is based on nucleases or chemicals that randomly cleave DNA at every phosphodiester bond
A bound regulatory protein blocks the phosphodiester bonds from attack, thereby revealing the proteins precise recognition site
The footprint is where no cleavage occurs

Question 13

What is the role of the EF hand?

Answer 13

Binds calcium or magnesium in structural signalling mdoe

Question 14

What does the SH2 domain bind?

Answer 14

Phosphoryated proteins

Question 15

What does the SH3 domain bind?

Answer 15

Proline rich motifs

Question 16

What does the PH domain bind?

Answer 16

Phosphorylated lipids

Question 17

What are the steps in the signalling pathway?

Answer 17

1) Activated receptor phosphorylates itself on tyrosine
2) It recruits insulin receptor substrate 1 via a PTB domain
3) The PH domain of IRSI binds to phosphoinositides on the plasma membrane
4) The activated rceptor phosphorylates IRSI on tyrosines
5) One phosphotyrosine binds the SH2 domain of GRB2 (adaptor protein)
6) GrB2 then uses two proleine rich SH3 domains to bind to two different proteins:
- SOS
- Scaffold protein

Question 18

How can we study protein protein interactions biochemically?

Answer 18

Centrifugation
Chromatography
Pulldown reactions

Question 19

How can we study protein protein interactions structurally?

Answer 19

X-ray crytallography
NMR
Electron Microscopy

Question 20

How do you tag proteins of interest?

Answer 20

Tag the protein of interest
eg use flag peptide (DYKDDDK)
Then use antibody against the tag to track the protein

Question 21

List two common tags for affinity chromatography/pull down

Answer 21

Glutathione -S-transferase (GST)

Hexahistidine (6xHis)

Question 22

List three common tags for immunoprecipitation

Answer 22

HA peptide
MyC peptide
Flag peptide

Question 23

What are the steps involved in GST affinity pulldown?

Answer 23

1) Protein X bound to GST coated beads
2) When cell extract is added interacting protein bind to protein X
3) Glutathione solution elutes the fusion protein together with the interacting protein

Question 24

What is the yeast two-hybrid screen?

Answer 24

Where bait and prey proteins are bound to DNA binding domains and transcriptional active domains
When 2 proteins interact the reporter gene is switched on

Question 25

What is ELISA?

Answer 25

Uses colour change and antibodies to test for a substance
Antigens from the sample are attached to the surface
A further antibody is applied over the surface so it can bind to an antigen - this antibody is linked to an enzyme
The enzyme substrate is then added